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AIMS: To investigate the usefulness of a straightforward filtration method for the isolation of Escherichia coli O157:H7 contaminants from milk for PCR detection. METHODS AND RESULTS: Escherichia coli O157:H7 is grown in milk and enriched in Luria-Bertani (LB) medium. Samples are filtered through a 0.45-microm pore membrane. The membrane is immersed in 200-microl lysis buffer and incubated at 95 degrees C for 10 min to release bacterial DNA for subsequent PCR detection. Under current conditions, the overall duration from filtration to PCR-ready DNA generation is <20 min, and the detection level for PCR was as low as 10 CFU of bacteria in 1 ml of milk. CONCLUSION: Bacterial contaminants of milk can be concentrated and isolated by a simple, one-step filtration and their DNA can be released for subsequent PCR detection by heating the filter membrane at 95 degrees C for 10 min. SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity of this method allows inexpensive, high throughput automation that meets the demands of modern food hygiene monitoring.

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The virD4 gene is one of the virulence genes present on the pTiC58 plasmid of Agrobacterium tumefaciens. Unexpectedly, we found that a pTi-free A. tumefaciens strain carried a protein of similar size to the plasmid-encoded VirD4 protein which reacted with VirD4-specific antibodies. This suggested that this strain may contain a homologue of the VirD4 protein. A chromosomal fragment encoding a protein of similar sequence to VirD4 was isolated and a 7.8 kilobase region surrounding the gene encoding this putative homologue was sequenced. This region contained four open reading frames, encoding putative proteins similar to proteins of known bacterial transfer and conjugation systems, viz., orf1 encoded a putative homologue of the TraA protein of the Rhizobium symbiosis plasmid pNGR234 and the TraA protein encoded by pTiC58 from A. tumefaciens plasmid pTiC58, orf3 encoded a protein very similar to the MobC protein encoded by the IncQ plasmid RSF1010 of E. coli and to MobS encoded by pTF1 from Thiobacillus ferrooxidans, whereas the predicted product of orf4 displayed similarity to the TraG protein encoded by the IncPalpha plasmid RP4 of E. coli, TraG and VirD4 encoded by A. tumefaciens plasmid pTiC58. The product of orf2 showed no significant similarity to any known protein. Preliminary assays with two orf4 mutants suggested that the product of this orf is involved in DNA transfer. The 7.8 kb chromosomal fragment seems to be closely related to the tra region of different conjugative plasmids and appears to be confined to Agrobacterium species, raising the question of the role of a chromosomal tra-like region during evolution.

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The T-pilus is a flexuous filamentous appendage that is essential for Agrobacterium tumefaciens virulence. T-pilus subunits are derived from a VirB2-processing reaction that generates cyclized polypeptide subunits. The T-pilus filament has a diameter of 10 nm and contains a lumen approximately 2 nm in diameter. Biogenesis of the T-pilus requires all 11 VirB proteins, but not the VirD4 protein, which is used in conjugal plasmid transfer. VirB4 and VirB11 are two ATPases that may form homohexameric rings within the transport apparatus, which is composed of VirB6-10 proteins.

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T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface. T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.

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Ros is a chromosomally-encoded repressor containing a novel C2H2 zinc finger in Agrobacterium tumefaciens. Ros regulates the expression of six virulence genes and an oncogene on the Ti plasmid. Constitutive expression of these genes occurs in the spontaneous mutant 4011R derived from the octopine strain Ach-5, resulting in T-DNA processing in the absence of induction, and in the biosynthesis of cytokinin. Interestingly, the mutation in 4011R is an Arg to Cys conversion at amino acid residue 125 near the C-terminus well outside the zinc finger of Ros. Yet, Ros bearing this mutation is unable to bind to the Ros-box and is unable to complement other ros mutants.

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The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells. Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined. In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation. We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation. Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A. tumefaciens strains used. With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A. tumefaciens rod-shaped cell in a polar manner. We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation. These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes. A search of the vir genes involved in controlling this biphasic reaction in induced A. tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction. The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A. tumefaciens.

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Pink disease of pineapple, caused by Pantoea citrea, is characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown chromogenic compounds. To elucidate the biochemical pathway leading to pink disease, we generated six coloration-defective mutants of P. citrea that were still able to oxidize glucose into gluconate. Three mutants were found to be affected in genes involved in the biogenesis of c-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate or 2-ketogluconate, resulting in an inability to produce the compound 2,5-diketogluconate (2,5-DKG). Thus, the production of 2,5-DKG by P. citrea appears to be responsible for the dark color characteristic of the pink disease of pineapple.

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The IncW plasmid pSa contains the gene ard encoding an antirestriction function that is specific for type I restriction and modification systems. The nucleotide sequence of ard was determined and an appropriate polypeptide of about 33 kDa was identified in Escherichia coli T7 expression system. Analysis of deduced amino acid sequence of Ard encoded by pSa revealed that this protein has no significant similarities with the known Ard proteins (ArdA and ArdB types) except the "antirestriction" motif (14 amino acid residues in length) conserved for all known Ard proteins. This finding suggests that pSa Ard may be classified as a new type of Ard proteins which we designated ArdC. The remarkable feature of ArdC is that it has a high degree of similarity (about 38 % identity) to the N-terminal region of RP4 TraC1 primase which includes about 300 amino acid residues and seems to be essential for binding to the single-stranded DNA and TraC1 protein transport to the recipient cells during the conjugal transfer of plasmid DNA. ArdC also binds to single-stranded DNA. In addition, this protein is able in vitro to protect the single-stranded but not double-stranded plasmid DNA against the activity of type II restriction endonuclease HhaI that cleaves both single and double-stranded DNA. We suggest that like TraC1, ArdC would be transported as a result of their interaction with the single-stranded DNA of transferred plasmid strand during conjugative passage through the cell envelope to the recipient bacterium. Such properties of ArdC protein might be useful to protect immediately the incoming single-stranded DNA from the host endonucleases.

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C2H2 zinc finger bearing proteins are a large superfamily of nucleic acid binding proteins, which constitute a major subset of eukaryotic transcription factors. Although originally thought to occur only in eukaryotes, a novel C2H2 zinc finger transcription factor, Ros, which regulates both prokaryotic and eukaryotic promoters has been found in bacteria. Phylogenically, Ros is distantly related to eukaryotic zinc finger regulators.

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Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.

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TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.

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When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens. Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium. We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A. tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression. When A. tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus. The extracellular complementation of an A. tumefaciens virE2 mutant (the T-DNA donor strain) by an A. tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain. These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A. tumefaciens. These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium. The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A. tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer. Overexpression of virB9, virB10, and virB11 in A. tumefaciens did not overcome oncogenic suppression by osa. The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.

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Pantoea citrea, the causal agent of pink disease of pineapple, harbors a cryptic plasmid of 5229 bp. designated pUCD5000. On the basis of nucleotide and amino acid sequence analyses, pUCD5000 contains both replication and mobilization loci (bom and mobCABD) that are similar to those in plasmids pSW100 and pSW200 in Pantoea stewartii and pEC3 in Erwinia carotovora subsp. carotovora. The survival of P. citrea on pineapple does not depend on pUCD5000. However, full pink coloration development, which is characteristic of the pink disease, appears to require this plasmid.

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Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P < or = 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::omega-Tc), confirmed the importance of ifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased beta-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.

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Virulence genes of Agrobacterium tumefaciens are under the control of positive and negative transcriptional regulators. We found that the transcriptional regulator Ros controls expression of the plant oncogene ipt, which encodes isopentenyl transferase, in A. tumefaciens. This enzyme is involved in biosynthesis of the plant growth hormone cytokinin in the host plant. An ipt promoter::cat reporter gene fusion showed a 10-fold increase in ipt promoter activity in A. tumefaciens ros mutant strains when compared with wild type. Also, increased levels (10- to 20-fold) of isopentenyl adenosine, the product of the reaction catalyzed by isopentenyl transferase, were detected in ros mutant strains. In vitro studies using purified Ros showed it binds directly to the ipt promoter. Analysis of the deduced Ros amino acid sequence identified a novel type of C2H2 zinc finger. In Ros the peptide loop spacing of the zinc finger is 9 amino acids as opposed to the invariant 12 amino acids in the classical C2H2 motif. Site-directed mutagenesis of Cys-82 and His-92 in this motif showed that these residues are essential for Zn2+ and DNA binding activities of Ros. The existence of such a regulator in Agrobacterium may be due to horizontal interkingdom retrotransfer of the ros gene from plant to bacteria.

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Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar.

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Previous studies have implicated the obligatory requirement for the vir regulon (or "virulon") of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19 degreesC as compared with 28 degreesC, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107-1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the "T pilus" of A. tumefaciens.

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Studies on the origin and evolution of plasmids may provide valuable insights on the promiscuous nature of DNA. The first examples of the selfish nature of nucleic acids are exemplified by primordial oligoribonucleotides which evolved into primitive replicons. The propagation of these molecules were likely patterned after the current viral RNA ribozymes, which have been recently shown to possess RNA synthesizing and template mediated polymerizing capabilities in the absence of proteins. The parasitic nature of nucleic acids is depicted by satellite nucleic acid molecules associated with viruses. The satellite of adenovirus and tobacco ringspot virus serve as established examples: they contain no open reading frames. Comparative analysis of the replication origins of virions and plasmids show them to be conserved, originating from the simplest autocatalytic replicon to highly complex and evolved plasmids, replicating by a rolling circle mechanism. The eventual association of proteins with nucleic acids provided added efficiency and protective advantages for molecular perpetuation. The promiscuous and selfish nature of plasmids is demonstrated by their ability to genetically engineer their host so that the host cell is best able to cope and survive in hostile environments. Survival of the host ensures survival of the plasmid. Sequestering of genes by plasmids occurs when the environmental conditions negatively affect the host. The sequestering mechanism is fundamental and forms the outreach mechanisms to generate and propagate macromolecules of increasing size when necessary for survival. The level of sophistication of plasmids increases with the addition of new genes such as those that allow the host to occupy a specific environment normally inhospitable to the host cell. The vast range of plasmid types which have obtained genes interchangeably reflect the levels of sophistication achieved by these macromolecules. The Ti plasmid in Agrobacterium tumefaciens and the pSym and accessory plasmids in Rhizobium illustrate the level of complexity attained by replicons.

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